Designation F2148 − 13 Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)1 This standard is issued under the fixed designation F2148; t[.]
Designation: F2148 − 13 Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)1 This standard is issued under the fixed designation F2148; the number immediately following the designation indicates the year of original adoption or, in the case of revision, the year of last revision A number in parentheses indicates the year of last reapproval A superscript epsilon (´) indicates an editorial change since the last revision or reapproval 1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use Scope 1.1 This practice provides a methodology to use an in-situ procedure for the evaluation of delayed contact hypersensitivity reactions 1.2 This practice is intended to provide an alternative to the use of guinea pigs for evaluation of the ability of a device material to stimulate delayed contact hypersensitivity reactions This alternative is particularly applicable for materials used in devices that contact only intact skin However, the guinea pig maximization test is still the recommended method when assessing the delayed hypersensitivity response to metals or when testing substances that not penetrate the skin but are used in devices that contact deep tissues or breached surfaces This practice may be used for testing metals, with the exception of nickel-containing metals, unless the unique physicochemical properties of the materials may interfere with the ability of LLNA to detect sensitizing substances 1.3 This practice consists of a protocol for assessing an increase in lymphocyte proliferation within the nodes draining the site of administration on the ears of mice 1.4 The LLNA has been validated only for low-molecularweight chemicals that can penetrate the skin The absorbed chemical or metabolite must bind to macromolecules, such as proteins, to form immunogenic conjugates 1.5 This practice is one of several developed for the assessment of the biocompatibility of materials Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application 1.6 Identification of a supplier of materials or reagents is for the convenience of the user and does not imply a single source Appropriate materials and reagents may be obtained from many commercial supply houses 1.7 The values stated in SI units are to be regarded as standard No other units of measurement are included in this standard Referenced Documents 2.1 ASTM Standards:2 F619 Practice for Extraction of Medical Plastics F720 Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices F750 Practice for Evaluating Material Extracts by Systemic Injection in the Mouse 2.2 Other Documents:3 ICCVAM NIH Publication No: 99-4494 The Murine Local Lymph Node Assay, 1999 ICCVAM NIH Publication NO: 11-7709 Usefulness and Limitations of the Murine Local Lymph Node Assay for Potency Categorization of Chemicals Causing Allergic Contact Dermatitis in Humans Terminology 3.1 Definitions: 3.1.1 AOO, n—acetone olive oil solution (4:1 v/v) is a suitable nonpolar solvent 3.1.2 aqueous solvent, n—in this assay refers to the polar solvent, saline 3.1.3 DMSO, n—dimethylsulfoxide (nonaqueous, suitable organic solvent) 3.1.4 DNCB, n—2,4-dinitrochlorobenzene 3.1.5 formalin, n—a 1⁄10 dilution of 37 to 39 % formaldehyde solution (formaldehyde) in PBS This practice is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee F04.16 on Biocompatibility Test Methods Current edition approved June 1, 2013 Published August 2013 Originally approved in 2001 Last previous edition approved in 2012 as F2148 – 07 (2012) DOI: 10.1520/F2148-13 For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org For Annual Book of ASTM Standards volume information, refer to the standard’s Document Summary page on the ASTM website Available from NICEATM, NIEHS, 79 Alexander Dr., Mail Drop EC-17, Research Triangle Park, NC 27709 Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959 United States F2148 − 13 6.3 Wholly aqueous solutions are not suitable for application to the ear Therefore, for use in the assay, add 0.05 g of hydroxyethyl cellulose4 to each 10 mL of the aqueous vehicle control and test solutions to aid in holding the solution to the ear One percent Pluronic L92 may also be used as an aqeuous vehicle 3.1.6 ICCVAM, n—Interagency Coordinating Committee on the Validation of Alternative Methods 3.1.7 nonaqueous solvent, n—in this assay refers to the organic or nonpolar solvent, which shall be dimethylsulfoxide (DMSO) or acetone olive oil (AOO) 3.1.8 PBS, n—phosphate buffered saline, pH 7.2 3.1.9 positive control, n—a substance capable of consistently stimulating lymphocyte proliferation 3.1.10 saline, n—0.9 % sodium chloride (aqueous, polar solvent) 3.1.11 TCA, n—5 % trichloroacetic acid 3.1.12 tritiated thymidine, n—H3methyl thymidine, specific activity Ci/mM (in PBS) I125 IUDR-radioactive uridine 3.1.13 vehicle controls, n—an aqueous, polar solvent and a non-aqueous, nonpolar solvent 6.4 The final specimen to be extracted should be prepared with a surface finish consistent with end-use application 6.5 The specimen shall be sterilized by the method to be used for the final product 6.6 Care should be taken that the specimens not become contaminated during preparation and aseptic technique is recommended Preparation of Positive Controls 7.1 Nonaqueous Positive Control—The use of a moderate positive control as a substitute or in addition to a strong positive control should be considered 7.1.1 Moderate Positive Control—Prepare a solution of 25 % hexyl cinnamic aldehyde (HCA) in an acetone:olive oil (4:1 v/v) solvent Shake the flask until a homogenous solution is obtained 7.1.2 Strong Positive Control—Weigh 0.025 g of DNCB and place in a flask Add enough DMSO to dissolve all of the DNCB Add more DMSO to bring the level up to 10 mL Cap and shake the flask until a homogenous solution is obtained 7.1.3 The dose level of the positive control should not produce systemic toxicity as evidenced by clinical observations Summary of Practice 4.1 Test and control substances or extracts are applied to the ears of test mice The draining lymph nodes are harvested and lymphocyte proliferation evaluated Comparisons are made with the control and test specimens tested under identical conditions Significance and Use 5.1 The propensity of a material to stimulate delayed contact hypersensitivity must be assessed before clinical application of devices containing this material Delayed hypersensitivity may occur anywhere in the body Systemic delayed hypersensitivity may have a complex set of reactions and consequences depending on the actual tissue/organ site of reaction Although the reactions are seldom life-threatening, severe tissue and organ damage my result over time Skin is the usual test site to determine the propensity of a material to cause delayed hypersensitivity 7.2 Aqueous Positive Control—Neutral buffered formalin is commercially available (Or dilute formaldehyde 1⁄10 in PBS Place mL of formaldehyde in a 10-mL flask Add enough PBS to mix the two solutions Add more PBS to bring the level up to 10 mL Cap and shake the flask until a homogeneous solution is obtained.) 5.2 The standard historical test methods have involved the use of guinea pigs with a cutaneous application and observation of the reaction site The use of the murine local lymph node assay results in a numerical quantitation of stimulation, rather than subjective evaluation and could be used to determine dose responses 7.3 Aqueous solutions are not suitable for application to the ear Therefore, for use in the assay, add 0.05 g of hydroxyethyl cellulose4 to each 10 mL of the aqueous positive control to aid in holding the solution to the ear until absorbed One percent Pluronic L92 may also be used as an aqueous vehicle 7.4 For all specimens requiring extractions, prepare an aqueous and non-aqueous extract (DMSO or AOO are recommended but other permissible extractants are listed in the ICCVAM documents) following the procedures described in Practice F619 5.3 This practice may not be predictive of events occurring during all types of implant applications The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748 Dosing of the Animals Preparation of Test Specimens 8.1 Healthy, non-pregnant female CBA/Ca or CBA/j mice that are seven to twelve weeks of age shall be used House the animals according to treatment group with five animals per cage 6.1 Specimens should be prepared in accordance with Practice F619 All solid materials shall be extracted Extractions shall be done with an aqueous (polar) solvent and a nonaqueous (nonpolar or organic) solvent, either DMSO or AOO 6.2 Liquid test articles and gels shall be used directly if they are not irritants A liquid that is an irritant shall be diluted with an aqueous or nonaqueous solvent based on solubility of the liquid test article until the solution is non-irritating “Final Report on the Safety Assessment of Hydroxyethylcellulose, Hydroxypropylcellulose, Methylcellulose, Hydroxypropyl Methylcellulose, and Cellulose Gum,” J Amer Coll Tox., Vol 5, No 3, 1986, pp 1-59 F2148 − 13 8.4.2 Confirm the concentration of this dilution Dilute 0.08 to 200 mL with water using a 200-mL flask Cap and mix well by inverting several times Remove two 1-mL samples and place in scintillation vials Add 10 mL of scintillation fluid to each vial, mix so that a vortex is formed, and “count” in a beta scintillation counter Count each vial three times and calculate the mean Calculate the concentration First convert counts per minute (cpm) to disintegrations per minute (dpm) as follows: 8.2 Day One—Uniquely identify each mouse (ear tags or ear notches may not be used) Weigh each mouse to the nearest whole gram 8.3 A minimum of five mice shall be used for each positive and negative control and each test sample They shall be treated daily for three consecutive days by topical application of 25 µL of one of the solutions to the dorsal surface of both ears For the aqueous groups only, the dorsal surface should be wiped with acetone just before treating to aid in absorption of the aqueous solution, although it will not be completely absorbed 8.3.1 For testing, other than liquid test articles, the groups shall include: aqueous and nonaqueous positive controls, aqueous and nonaqueous vehicle controls, aqueous extract of the test sample, and nonaqueous extract of test sample 8.3.2 For testing of liquid test articles, the groups shall include: aqueous and nonaqueous positive controls, the liquid test sample, and either an aqueous or a nonaqueous vehicle control appropriate for the nature of the liquid sample 8.3.3 The extract shall be used within 24 h of preparation The extract should be stored in a stoppered container at room temperature The applications shall be performed at 24 h intervals on Days and Table describes the events for each day of the test 8.3.4 Observe each mouse daily for signs of local irritation at the application site and for signs of systemic toxicity (see Practices F720 and F750) It may be advisable to pretest two mice if it is suspected that the material may be an irritant cpm dpm decimal counter efficiency For verification of the working tritiated thymidine solution, determine the closeness of the concentration to 80 µCi/mL The final diluted solution contains 0.032 µCi Since 1.0 µCi = 220 000 dpm, then 0.032 µCi = 71 040 dpm Therefore: the µCi of the working solution Make adjustments to the solution as needed Make similar verifications if I125 IUDR is used 8.5 In-Situ Labeling (Day 6)—(72 h after the last treatment was applied to the ears) 8.5.1 Record the weight of the mouse to the nearest gram Inject the mouse intravenously with 250-µL sterile PBS containing 20 µCi of tritiated thymidine or µCi I125IUDR via the lateral tail vein using a 1.0-mL syringe and a needle no larger than 25 gage The tail veins may be dilated for easier intravenous injection by placing the mice under a heat lamp NOTE 1—The following steps through 9.3.3 until precipitation for 18 h take more than h to complete and the laboratory needs to be prepared to accommodate this Lymph Node Collection and Lymph Node Cell Preparation 8.4 Radiolabeled Tracer Preparation—Prepare tritiated thymidine to a working concentration of 80 µCi/mL (v/v) The use of I125 I-UDR at µCi/mL in PBS 10-5 M fluorodeoxyuridine is also acceptable Each mouse will receive 250 µL of this All standard precautions associated with using radioactive materials shall be adhered to The laboratory shall be licensed to use radioactive material and all personnel shall be appropriately trained and certified 8.4.1 To prepare the tritiated thymidine solution, add 0.8 mL of 1.0-mCi/mL tritiated thymidine (specific activity 2.0 Ci/ mM) to a stoppered flask Add sterile PBS to make 10 mL Cap and mix well NOTE 2—All equipment and solutions from this point should be treated as radioactive with appropriate precautions NOTE 3—If the investigators are not familiar with the location of the lymph nodes, refer to the diagram in the ICCVAM documents, consult a mouse anatomy book, or use a dye in trial mice to learn to locate the appropriate nodes One suggested procedure is to inject 0.1 mL of % Evan’s blue dye intradermally into the tissue of the ear and then euthanize the mice after to 10 Dissect the mice to expose the nodes 9.1 Euthanize the mice h 45 after injection of the radioisotope 9.2 Excise the draining (auricular) lymph node of each ear 9.3 Prepare a single-cell suspension of lymph node cells (LNC) for each mouse 9.3.1 Pool the nodes from both the left and right side of a single mouse in a labeled test tube containing to mL of PBS Snip the nodes from a single mouse into small pieces with small scissors or transfer the lymph nodes directly onto a 200-mesh screen/cell dissociation cup Gently mash the pieces through the screen directly into a 15-mL tube Wash the mesh with to mL of PBS to facilitate the transfer of cell debris into the tube Bring the volume in the centrifuge tube up to 10 mL 9.3.2 Repeat this procedure for each animal 9.3.3 Centrifuge the samples for 10 at 190 to 200 xg at to 8°C Remove each supernatant by aspiration, leaving to mL of supernatant above each pellet Gently agitate each pellet then bring up to 10 mL with PBS and resuspend by TABLE LLNA Timetable Day Activity